Use your antibodies-online credentials, if available.
No Products on your Comparison List.
Your basket is empty.
Find out more
Get this product for free
Bring all reagents to room temperature before use8.1.Assay Design Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running zeros and standards. Each sample, standard and zero should be tested in duplicate. Remove sufficient Microwell Strips for testing from the pouch immediately prior to use. Return any wells not required for this assay with desiccant to the pouch. Seal tightly and return to 2-8 °C storage.8.2.Preparation of Wash Buffer Dilute the (200x) wash buffer concentrate 200 fold with distilled water to give a 1x working solution. Pour entire contents (10 mL) of the Washing Buffer Concentrate into a clean 2,000 mLgraduated cylinder. Bring final volume to 2,000 mLwith glass-distilled or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2°-8 °C for up to 1 week.8.3.Preparation of Standard Diluent Buffer Add the contents of the vial (10x concentrate) to 225 mL of distilled water before use. This solution can be stored at 2-8 °C for up to 1 week.8.4.Preparation of Standard Standard vials must be reconstituted with the volume of standard diluent shown on the vial immediately prior to use. This reconstitution gives a stock solution of 38U/mL of CD86. Mix the reconstituted standard gently by inversion only. Serial dilutions of the standard are made directly in the assay plate to provide the concentration range from 38 to 1.2Ug/mL. A fresh standard curve should be produced for each new assay. Immediately after reconstitution add 200 μL of the reconstituted standard to wells A1 and A2, which provides the highest concentration standard at 38U/mL Add 100 μL of Standard Diluent to the remaining standard wells B1 and B2 to F1 and F2 Transfer 100 μL from wells A1 and A2 to B1 and B2. Mix the well contents by repeated aspirations and ejections taking care not to scratch the inner surface of the wells Continue this 1:1 dilution using 100 μL from wells B1 and B2 through to wells F1 and F2 providing a serial diluted standard curve ranging from 38 to 1.2U/mL Discard 100 μL from the final wells of the standard curve (F1 and F2) Alternatively these dilutions can be performed in separate clean tubes and immediately transferred directly into the relevant wells.8.5.Preparation of Samples Before testing, serum or plasmas samples have to be diluted 1:20 in Standard Diluent (buffer).8.6.Preparation of Biotinylated anti-CD86 It is recommended this reagent is prepared immediately before use. Dilute the biotinylated anti-CD86 with the biotinylated antibody diluent in an appropriate clean glass vial using volumes appropriate to the number of required wells.8.7.Preparation of Streptavidin-HRP It is recommended to centrifuge vial for a few seconds in a microcentrifuge to collect all the volume at the bottom. Dilute the 5 μL vial with 0.5 mL of HRP diluent immediately before use. Do-not keep this diluted vial for future experiments. Further dilute the HRP solution to volumes appropriate for the number of required wells in a clean glass vial.
Cell culture supernatants, serum, plasma or other biological samples will be suitable for use in the assay Remove serum from the clot or red cells, respectively, as soon as possible after clotting and separation. Cell culture supernatants: Remove particulates and aggregates by spinning at approximately 1000 x g for 10 min Serum: Avoid any unintentional stimulation of the cells by the procedure Use pyrogen/endotoxin free collecting tubes. Serum should be removed rapidly and carefully from the red cells after clothing. For that, after clothing, centrifuge at approximately 1000 x g for 10 min and remove serum. Plasma: EDTA, citrate and heparin plasma can be assayed Spin samples at 1000 x g for 30 min to remove particulates. Harvest plasma. Storage: If not analyzed shortly after collection, samples should be aliquoted (250-500 μL) to avoid repeated freeze-thaw cycles and stored frozen at -70 °C Avoid multiple freeze-thaw cycles of frozen specimens. Recommendation: Do not thaw by heating at 37 °C or 56 °C. Thaw at room temperature and make sure that sample is completely thawed and homogeneous before use. When possible avoid use of badly haemolysed or lipemic sera. If large amounts of particles are present these should be removed prior to use by centrifugation or filtration.
We strongly recommend that every vial is mixed thoroughly without foaming prior to use except the standard vial which must be mixed gently by inversion only. Prepare all reagents as shown in section8.Note: Final preparation of Biotinylated anti-CD86 and Streptavidin-HRP should occur immediately before use. 1.Addition Prepare Standard curve as shown in section 8.42.Addition Add 100 μL of each standard, sample and zero (Standard Diluent) in duplicate to appropriate number of wells3.Addition Add 50 μL of diluted biotinylated anti-CD86 to all wells4.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 2 hours5.Wash Remove the cover and wash the plate as follows: a) Aspirate the liquid from each well b) Dispense 0.3 mLof 1x washing solution into each well c) Aspirate the contents of each well d) Repeat step b and c another two times6.Addition Add 100 μL of Streptavidin-HRP solution into all wells7.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 30 min8.Wash Repeat wash step5.9. Addition Add 100 μL of ready-to-use TMB Substrate Solution into all wells10.Incubation Incubate in the dark for 10-20 minutes at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil11.Addition Add 100 μL of H2SO4:Stop Reagent into all wells Read the absorbance value of each well (immediately after step 11.) on a spectrophotometer using 450 nm as the primary wavelength and optionally 630 nm as the reference wave length (610 nm to 650 nm is acceptable). Incubation time of the substrate solution is usually determined by the ELISA reader performance. Many ELISA readers only record absorbance up to 2.0 O.D. Therefore the colour development within individual microwells must be observed by the analyst, and the substrate reaction stopped before positive wells are no longer within recordable range
Calculate the average absorbance values for each set of duplicate standards and samples. Ideally duplicates should be within 20 % of the mean. Generate a linear standard curve by plotting the average absorbance of each standard on the vertical axis versus the corresponding human CD86 standard concentration on the horizontal axis. The amount of CD86 in each sample is determined by extrapolating OD values against CD86 standard concentrations using the standard curve.Every laboratory must produce a standard curve for each set of microwell strips assayed. Do not extrapolate the standard curve beyond the maximum standard curve point. The dose-response is non-linear in this region and good accuracy is difficult to obtain. Concentrated samples above the maximum standard concentration must be diluted with Standard diluent or with your own sample buffer to produce an OD value within the range of the standard curve. Following analysis of such samples always multiply results by the appropriate dilution factor to produce actual final concentration. The influence of various drugs on end results has not been investigated. Bacterial or fungal contamination and laboratory cross-contamination may also cause irregular results. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Washing Buffer, fill with Washing Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. As with most biological assays conditions may vary from assay to assay therefore a fresh standard curve must be prepared and run for every assay.